Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1)Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Conclusion: Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs;(2)Cancer heterogeneity is dynamically regulated by genetic, epigenetic and cellular microenvironmental factors. These factors also play important roles in transcriptional reprogramming and cell fate changes in cancer cells. DNA methylation constitute an important part of the epigenetic mechanism modulating cancer and stem cells. We have studied the interaction between epigenetic alterations and local microenvironment in regulation of cancer stem cell (CSC) properties using Zebularine (Zeb), a DNA-methyltransferase1 (DNMT1) inhibitor, in combination with the modulation of cell density in culture. Seven human hepatoma cell lines, including two early passage cell lines established from primary HCC, were plated at high (HD) and low density (LD), and exposed to 100 microM of Zeb for 3 days. Thereafter, cells were dissociated and plated at clonal density over several passages (generation G1 to G5) in drug-free, serum-free, and adherent-free conditions. The differences in self-renewal, gene expression, tumorigenicity and metastatic potential of cells derived from G1 to G5 spheres were examined. shDNMT1-Huh7 cell line was generated to address the role of DNMT1 in long-term self-renewal. Results: The transient exposure to Zeb produced differential cell density-depended responses in 5/7 tested HCC cell lines. In cell lines which were scored as Zeb-sensitive, a 3-day drug pre-treatment of LD cultures (LDZ) caused a remarkable increase in primary sphere formation. This effect persisted through at least five subsequent sphere generations in methylcellulose sphere-forming assays in drug- and serum-free medium. In striking contrast, untreated LD cells failed to form primary spheres while the sphere forming potential of HD and HD Zeb treated (HDZ) cells rapidly decreased over the first three generations. The depletion of DNMT1 in Huh7, a Zeb-sensitive cell line, by a stable lentiviral transduction of DNMT1 shRNA similarly increased the self-renewal potential of LD cells, providing evidence that the density-dependent effect was epigenetically regulated. Conversely, the phenotypic response of Zeb-resistant WRL68 and HepG2 was independent of drug and plating schema despite a complete depletion of DNMT1 protein.The increase in sphere formation in LDZ cells strongly correlated with a stable overexpression of CSC-related markers (CD133, CD44, EpCAM), as well as key genes involved in ESC self-renewal (Bmi1, alapha-SUZ12) and epithelial-mesenchymal transition (EMT) (beta-catenin, ZEB1, VIM, SNAL1). Likewise, when LDZ, HD and HDZ spheres were dissociated and injected s.c. into NOD/SCID mice (100 cells/injection site), the LDZ cells generated tumors more rapidly and with higher penetrance, and showed more frequent brain and lung metastasis. Both gene reactivation and tumorigenicity progressively increased G1 to G4. Tumors derived from G1-G4 LDZ cells were also increasingly more vascular. Global transcriptome analysis of LDZ spheres at G1-G4 confirmed that a common LDZ signature was characterized by genes associated with oncogenic signaling pathways including tumor initiation, metastasis, and vasculogenesis and was able to predict clinical outcome of liver cancer patients. We conclude that epigenetic reprogramming of liver cancer cell lines induced by a combined modulation of DNA methylation and cellular microenviroment can enhance and stabilize CSC properties;(3)Classification of human liver cancer into biologically distinct subgroups suggests its origin from different hepatic lineage cells. To clarify the contribution of the lineage stage in liver oncogenesis, we transduced H-Ras/SV40 large T into hepatic progenitor cells (HCP), hepatoblasts (HB) and terminally differentiated adult hepatocytes (AH). Regardless of origin, the transformed cell types acquired common cancer stem cell traits both in vitro and in vivo. However, expression analyses distinguished tumors from different lineage stages demonstrating that distinct genetic changes occur during malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. Our results demonstrate that any hepatic lineage cell can be a target population for transformation via activation of diverse cell-specific pathways. Primary human liver cancer (PLC) is the third most lethal cancer worldwide with incidence rising in United States and Western Europe. PLC can arise from liver epithelial cell lineages and adult hepatic progenitor cells. Identification of cells that are susceptible to oncogenic transformation is critical for both diagnosis and treatment. Employing a mosaic mouse model of PLC we demonstrated that any cell within the hepatic lineage can be a target of malignant transformation and display a cancer stem cell mode of tumorigenesis. The identification of common and cell of origin specific phenotypic and genetic changes should provide novel therapeutic targets for the treatment of PLC. In particular, our demonstration of the general role of c-myc in PLC oncogenesis offers unique therapeutic opportunities.

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National Cancer Institute (NCI)
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Marquardt, Jens U; Thorgeirsson, Snorri S (2014) SnapShot: Hepatocellular carcinoma. Cancer Cell 25:550.e1
Raggi, Chiara; Factor, Valentina M; Seo, Daekwan et al. (2014) Epigenetic reprogramming modulates malignant properties of human liver cancer. Hepatology 59:2251-62
Marquardt, Jens U; Thorgeirsson, Snorri S (2013) Sall4 in ""stemness""-driven hepatocarcinogenesis. N Engl J Med 368:2316-8
Holczbauer, Ágnes; Factor, Valentina M; Andersen, Jesper B et al. (2013) Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types. Gastroenterology 145:221-231
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Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E et al. (2012) Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division. Stem Cells 30:591-8
Marquardt, Jens U; Raggi, Chiara; Andersen, Jesper B et al. (2011) Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways. Hepatology 54:1031-42
Woo, Hyun Goo; Wang, Xin Wei; Budhu, Anuradha et al. (2011) Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma. Gastroenterology 140:1063-70

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